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Figure 1.4.12.2 Cyclopentanoperhydrophenanthrene.

Extensive studies of the structure-activity relationships of anabolic/androgenic steroids have demonstrated that the following structural attributes are necessary for maximal andro-genic and anabolic effects: rings A and B must be in the trans configuration;3 hydroxy function at C17 must be in the £ conformational state;5,6 and high electron density must be present in the area of C2 and C3.7 The presence of a keto or hydroxl group at position 3 in the A-ring usually enhances androgenic and anabolic activity, but it is not absolutely necessary for these effects.7 A few examples of structural alterations that enhance anabolic activity include: removal of the C-19 methyl group;8 methyl groups at the 2a and 7a positions;9'10 a flourine at the 9a position; or a chlorine at the 4a position.10,11 To make it easier to visualize where these modifications are made in the ring structure, a numbered steroid skeletal ring structure, namely the cyclopentanoperhydrophenanthrene ring, is shown in Figure 1.4.12.2.

It is essential to understand that structure-activity analysis can only predict whether or not a steroid is likely to produce androgenic/anabolic effects. It then becomes necessary to examine the steroid in the laboratory to determine whether the prediction is, in fact, true. It is also important to note that numerous studies performed over the years and designed to separate androgenic activity from anabolic activity have failed to obtain such a separation of pharmacological effect. That is, steroids found to possess androgenic activity also have anabolic activity and vice versa. An examination of the scientific and medical literature reveals that there are, indeed, additional steroids that are not specifically listed in the law but which do, based upon available data, probably produce androgenic/anabolic effects. A listing of some of these steroids is provided below.

Androisoaxazole

Bolandiol

Bolasterone

Bolenol

Flurazebol

Mebolazine

Mesabolone

Mestanolone

Methyltrienolone

Norbolethone

Norclostebol

Oxabolone Cypionate

Quinbolone

Stenbolone

1.4.12.3 Forensic Analysis

For the forensic chemist, when a steroid is tentatively identified, an additional problem arises, namely obtaining an analytical standard. Many products found in the illicit U.S. market are commercially available only outside of the U.S. Locating and making contact with a foreign distributor is one problem. Requesting and then receiving a legitimate standard is another problem. The expense incurred in obtaining these standards can be quite high. Once the standard has been received, authentication then enters the analytical process. If a primary standard is unavailable, an optimized analytical process presents a real problem. Fortunately, most steroids received by forensic science laboratories are labeled directly or have labeled packaging. So a manufacturer can be identified, and there is a starting point for the chemist in confirming the material as a particular steroid.

There are no known color tests, crystal tests, or TLC methods which are specific to anabolic steroids. Screening can be accomplished by GLC or HPLC. GLC sometimes presents a problem because of thermal decomposition in the injection port thereby resulting in several peaks. The steroid will not always be the largest peak. On-column injection will usually solve this problem. However, oil-base steroids rapidly foul or degrade GC columns. Samples in oils can be extracted with methanol/water 9:1 prior to injection onto a GC. Retention times for some anabolic steroids are quite long and nearly triple or quadruple that of heroin. Recognizing that several anabolic steroids are readily oxidized in polar, protic solvents vs. halogenated hydrocarbons, screening and analysis must be accomplished as soon as possible after isolation and dilution.

GC/MS does provide definitive spectra; however, different MS systems may provide differences in the spectra for the same steroid. These differences can be traced to the quality of the MS source and the injection liner, thermal decomposition products, and induced hydration reactions related to high source temperatures set by the MS. C13NMR is the most rigorous identification technique. The limitation here is the need for pure samples and high sample concentrations. Identification by infrared alone can result in problems due to polymorphism. This can be minimized by ensuring that the sample and standard are recrystallized from the same solvent.

Ideally, all anabolic steroids should be identified using two analytical methodologies which yield the same conclusions. The collection of a library of analytical data on different anabolic steroids is essential for the subsequent identification of steroids sent to the laboratory. An ability to interpret mass spectral data will be important in making an identification in so far as determining a molecular formula. Interpreting NMR data will be important in determining how substitutents are attached to the parent steroid ring structure.

It should be noted that selected steroids, such as testosterone, nandrolone, methenolone, boldenone, methandriol, and trenbolone, will often be encountered by the laboratory, not as the parent drug, but instead as an ester. The type of ester will be dependent upon the particular steroid. For example, nandrolone is primarily found as a decanoate, laurate, or phenpropionate ester. Testosterone, although it is found as a parent drug, is actually most commonly encountered as the propionate, enanthate, cypionate, decanoate, isocaproate, or undecanoate esters. Less commonly encountered testosterone esters include the acetate, valerate, and undecylenate esters. Methenolone is almost always found in either the acetate or enanthate esterified form.

Upon reaching the forensic science laboratory, steroid preparations will be handled differently depending on the way each preparation is formulated. Tablets can be handled by finely grinding and extracting with chloroform or methanol. Aqueous suspensions can be handled by dilution/solution with methanol for HPLC screening or by extraction with chloroform for GC screening. Oils require a more specialized extraction which is outlined below:

1. 1 ml of oil is mixed with 10 mls of methanol/water 9:1 and the mixture is allowed to sit overnight at 0°C.

2. Methanol water mixture is removed by evaporating to dryness under a stream of nitrogen at 60°C.

3. The resulting solid is subjected directly to an IR analysis or taken up in an appropriate solvent for MS or NMR analysis.

4. Exhibits containing mixtures of anabolic steroids require semi-prep scale HPLC for rigorous isolation and identification.

5. Isocratic or gradient HPLC is recommended for quantitation of anabolic steroids.

What steroids have been the most predominate in the United States in the past few years? From January 1990 to October 1994, the following steroids or their esters have been identified by DEA laboratories.

This list provides an objective evaluation of what this chemist has encountered in the not too distant past. The data on these particular steroids should form the basis of a reference collection for comparison with future submissions.

Steroids or esters of a steroid

Numbers of Cases Exhibits

Testosterone

Nandrolone

Methenolone

Methandrostenolone

Oxymetholone

Stanozolol

Fluoxymesterone

Methyltestosterone

Boldenone

Mesterolone

Oxandrolone

Trenbolone

Methandriol

Drostanolone

Mibolerone

Stanolone

Testolactone

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